Transferases (EC 2) catalyze transfer of a functional group from one substance to another. Glycosyltransferases, a superfamily of enzymes, are involved in synthesizing the carbohydrate portions of glycoproteins, glycolipids and glycosaminoglycans. Specific glycosyltransferases synthesize oligosaccharides by the sequential transfer of the monosaccharide moiety of an activated sugar donor to an acceptor molecule. Hence, a “glycosyltransferase” catalyzes the transfer of a sugar moiety from its nucleotide donor to an acceptor moiety of a polypeptide, lipid, glycoprotein or glycolipid. This process is also known as “glycosylation”. A carbohydrate portion which is structural part of e.g. a glycoprotein is also refered to as “glycan”. Glycans constitute the most prevalent of all known post-translational protein modifications. Glycans are involved in a wide array of biological recognition processes as diverse as adhesion, immune response, neural cell migration and axonal extension. As structural part of glycoproteins glycans also have a role in protein folding and the support of protein stability and biological activity.
In glycosyltransferase catalysis, the monosaccharide units glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), galacturonic acid (GalUA) and xylose are activated as uridine diphosphate (UDP)-α-D derivatives; arabinose is activated as a UDP-β-L derivative; mannose (Man) and fucose are activated as GDP-a-D and GDP-β-L derivatives, respectively; and sialic acid (=Neu5Ac; =SA) is activated as a CMP derivative of β-D-Neu5Ac.
Many different glycosyltransferases contribute to the synthesis of glycans. The structural diversity of carbohydrate portions of glycoproteins is particularly large and is determined by complex biosynthetic pathways. In eukaryotes the biosynthesis of the glycan-part of glycoproteins takes place in the lumen of the endoplasmatic reticulum (“ER”) and the Golgi apparatus. A single (branched or linear) carbohydrate chain of a glycoprotein is typically a N- or an O-linked glycan. During post-translational processing, carbohydrates are typically connected to the polypeptide via asparagine (“N-linked glycosylation”), or via serine or threonine (“O-linked glycosylation”). Synthesis of a glycan, no matter whether N- or O-linked (=“N-/O-linked”) is effected by the activity of several different membrane-anchored glycosyltransferases. A glycoprotein may comprise one or more glycan-connected amino acids (=“glycosylation sites”). A specific glycan structure may be linear or branched. Branching is a notable feature of carbohydrates which is in contrast to the linear nature typical for DNA, RNA, and polypeptides. Combined with the large heterogeneity of their basic building blocks, the monosaccharides, glycan structures exhibit high diversity. Furthermore, in members of a particular glycoprotein species the structure of a glycan attached to a particular glycosylation site may vary, thus resulting in microheterogeneity of the respective glycoprotein species, i.e. in a species sharing the same amino acid sequence of the poypeptide portion.
A sialyltransferase (=“ST”) is a glycosyltransferase that catalyzes transfer of a sialic acid (=5-N-acetylneuramic acid=Neu5Ac=NANA) residue from a donor compound to (i) a terminal monosaccharide acceptor group of a glycolipid or a ganglioside, or (ii) to a terminal monosaccharide acceptor group of an N-/O-linked glycan of a glycoprotein. For mammalian sialyltransferases including human ST species there is a common donor compound which is cytidine-5′-monophospho-N-acetylneuraminic acid (=CMP-Neu5Ac=CMP-NANA). Transfer of a sialic acid residue is also referred to as “sialylating” and “sialylation”.
In the glycan structure of a sialylated glycoprotein the (one or more) sialyl moiety (moieties) is (are) usually found in terminal position of the oligosaccharide. Owing to the terminal, i.e. exposed position, sialic acid can participate in many different biological recognition phenomena and serve in different kinds of biological interactions. In a glycoprotein more than one sialylation site may be present, i.e. a site capable of serving as a substrate for a sialyltransferase and being an acceptor group suitable for the transfer of a sialic acid residue. Such more than one site can in principle be the termini of a plurality of linear glycan portions anchored at different glycosylation sites of the glycoprotein. Additionally, a branched glycan may have a plurality of sites where sialylation can occur.
According to current knowledge, a terminal sialic acid residue can be found (i) α2→3 (α2,3) linked to galactosyl-R, (ii) α→26 (α2,6) linked to galactosyl-R, (iii) α2→6 (α2,6) linked to N-acetylgalactosaminidyl-R, (iv) α2→6 (α2,6) linked to N-acetylglucosaminidyl-R, and (v) α2→8/9 (α2,8/9) linked to sialidyl-R, wherein -R denotes the rest of the acceptor substrate moiety. Hence, a sialyltransferase active in the biosynthesis of sialylconjugates (=“sialylation”) is generally named and classified according to its respective monosaccharide acceptor substrate and according to the 3, 6 or 8/9 position of the glycosidic bond it catalyzes. Accordingly, in the literature known to the art, e.g. in Patel R Y, et al, Glycobiology 16 (2006) 108-116, reference to eukaryotic sialyltransferases is made such as (i) ST3Gal, (ii) ST6Gal, (iii) ST6GalNAc, or (v) ST8Sia, depending on the hydroxyl position of the acceptor sugar residue to which the Neu5Ac residue is transferred while forming a glycosidic bond . Reference to sialyltransferases in a more generic way can also be made e.g. as ST3, ST6, ST8; thus, “ST6” specifically encompasses the sialyltransferases catalyzing an α2,6 sialylation.
The disaccharide moiety β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine (=Galβ1,4GlcNAc) is a frequent terminal residue of the antennae of N-linked glycans of glycoproteins, but may be also present in O-linked glycans and in glycolipids. The enzyme β-galactoside-α2,6-sialyltransferase (=“ST6Gal”) is able to catalyze α2,6-sialylation of a terminal Galβ1,4GlcNAc of a glycan or a branch of a glycan (=“antenna”). For general aspects thereof, reference is made to the document of DallOlio F. Glycoconjugate Journal 17 (2000) 669-676. In human and in other mammals there appear to be several species of ST6Gal. The present disclosure particularly deals with human β-galactoside-α-2,6-sialyltransferase I (=hST6Gal-I; EC 2.4.99.1 according to IUBMB Enzyme Nomenclature), but is not limited thereto.
The ST6 group of sialyltransferases comprises 2 subgroups, ST6Gal and ST6GalNAc. The activity of ST6Gal enzymes catalyzes transfer of a Neu5Ac residue to the C6 hydroxyl group of a free galactosyl residue being part of terminal Galβ1,4GlcNAc in a glycan or an antenna of a glycan, thereby forming in the glycan a terminal sialic acid residue α2→6 linked to the galactosyl residue of the Galβ1,4GlcNAc moiety. The resulting newly formed terminal moiety in the glycan is Neu5Acα2,6Galβ1,4GlcNAc.
The wild-type polypeptide of human β-galactoside-α-2,6-sialyltransferase I (hST6Gal-I) at the time of filing of the present document was disclosed as “UniProtKB/Swiss-Prot: P15907.1” in the publically accessible NCBI database (www.ncbi.nlm.nih.gov/protein/115445). Further information including coding sequences are provided as hyperlinks compiled within the database entry “Gene ID: 6480” (www.ncbi.nlm.nih.gov/gene/6480).
Mammalian sialyltransferases share with other mammalian Golgi-resident glycosyltransferases a so-called “type II architecture” with (i) a short cytoplasmic N-terminal tail, (ii) a transmembrane fragment followed by (iii) a stem region of variable length and (iv) a C-terminal catalytic domain facing the lumen of the Golgi apparatus (Donadio S. et al. in Biochimie 85 (2003) 311-321). Mammalian sialyltransferases appear to display significant sequence homology in their catalytic domain.
Donadio S. et al. expressed several N-terminally truncated variants of hST6Gal-I in CHO cells and found that N-terminal deletions comprising the first 35, 48, 60 and 89 amino acids yielded mutant enzymes which nevertheless were still active in transferring sialic acid to exogenous acceptors.
Glycosylation is an important posttranslational modification of proteins influencing protein folding, stability and regulation of the biological activity. The sialyl mojety (=sialic acid, 5-N-acetylneuramic acid, Neu5Ac) is usually exposed at the terminal position of N-glycosylation and therefore, a major contributor to biological recognition and ligand function, e.g. IgG featuring terminal sialic acids were shown to induce less inflammatory response and increased serum half-life.
The use of glycosyltransferases for enzymatic synthesis of defined glycan structures is becoming a tool to direct N-glycosylation of therapeutic proteins such as antibodies. Since glycosyltransferases of prokaryotic origin usually do not act on complex glycoprotein structures, sialyltransferases of mammalian origin are preferred. For example, Barb et al. (2009) prepared highly potent sialylated forms of the Fc fragment of immunoglobulin G using isolated human ST6Gal-I. However, the access to recombinant ST6Gal-I for therapeutic applications is still limited due to low expression and/or poor activity in various hosts (Pichia pastoris, Spodoptera frugiperda and E. coli).
It is known to the art that mammalian glycosyltransferases can be used advantageously for in vitro sialylating a complex target molecule such as a glycoprotein or a glycolipid. However, the opposite reaction (sialidase activity, hydrolytic cleavage of a terminal sialyl residue from a glycan moiety) is typically provided by a neuraminidase. The original finding by the present inventors is, however, that a variant of a sialyltransferase of mammalian origin displays sialidase activity. In fact, a specific variant of human human β-galactoside-α-2,6-sialyltransferase I with an N-terminal truncation can be used for both, (i) sialylation of a target glycoprotein and (ii) hydrolytic cleavage of sialyl residues from the sialylated target glycoprotein. Depending on the control of kinetics of the variant enzyme, sialylation can be controlled quantitatively. That is to say, the present disclosure provides means, methods and conditions allowing to sialylate just one out of the several acceptor sites as opposed to sialylating two or more, or even all acceptor sites of the target molecule.
This paves the way for a number of different approaches, particularly in the field of in vitro glycoengineering of immunoglobulins, and also of other glycosylated target molecules. Here specifically and exemplarily a method is provided resulting in the production of predominantly mono-sialylated or bi-sialylated immunoglobulin G molecules. However, a number of other in vitro sialylation approaches with quantitative sialylation control of the target molecule to be sialylated become feasible and can be deduced from the present disclosure.
In a specific embodiment this document further discloses the high-yield expression of a Δ89 N-terminal truncation variant of human β-galactoside-α-2,6-sialyltransferase I (hST6Gal-I, EC 2.4.99.1; data base entry P15907) by transient gene expression in HEK293 cells with yields up to 100 mg/L featuring a surprisingly distinct sialylation activity.